Novel coupling agents, the active intermediates and the conjugates thereof and the use of said conjugates in diagnostic methods

ABSTRACT

The present invention concerns coupling agents having the following general formula:  
                 
activated intermediates consisting of a coupling agent such as defined above with either a molecule having on its surface at least one aldehyde and/or ketone function before conjugation, or with one to eight molecules having on their surface at least one free thiol function before conjugation, conjugates consisting of a coupling agent such as defined above, of a molecule having on its surface at least one aldehyde and/or ketone function and of one to eight molecules having on their surface at least one free thiol function, and the use of said conjugates for in vitro diagnostic methods of diseases involving recognition of a ligand-anti-ligand pair.

The present invention concerns heterobifunctional coupling agents whichmay be used in particular for the coupling of molecules having on theirsurface at least one aldehyde and/or ketone function and moleculeshaving on their surface at least one free thiol function.

Coupling agents are useful in the area of diagnosis, in particular todetect reactions of ligand-anti-ligand type such as antigen-antibody orprotein-protein, since they allow the direct coupling of a molecule ofbiological interest, such as an antigen, with a detecting molecule suchas an enzyme. The binding of the molecule of biological interest toanother molecule such as an antibody is evidenced by means of thedetecting molecule.

For this purpose, coupling agents must be bifunctional having both achemical function enabling their coupling with said molecule ofbiological interest and a further chemical function enabling theircoupling with said detecting molecule, and must also have a spacer armallowing sufficient distancing of the coupled molecules.

Various heterobifunctional coupling agents are known from the prior art.These coupling agents, found in the PIERCE catalogue for example,essentially have a maleimide function, a hydrazone function and a spacerarm of saturated or aromatic alkyl type. These coupling agents are usedfor coupling molecules having free thiol functions and also for couplingmolecules having aldehyde or ketone functions. However, these couplingagents have the disadvantage of being scarcely soluble in aqueous mediain particular on account of the hydrophobic nature of the spacer arm,and often require the use of organic co-solvents when used, inparticular to prepare conjugates. In addition, the proteins coupled tothese coupling agents have a tendency to precipitate during marking.

Mikolajczyk S. et al. (Bioconjugate Chemical, 1994, 5(6), 636-646)describe the use of a coupling agent having a maleimide function, anaminooxyalkylene function and a relatively short spacer arm containingL-lysine in particular. This coupling agent is used for coupling twomodified proteins, β-lactamase and the Fab murine peptide fragment. Thedisadvantages of this coupling agent are that it has a short spacer armnot allowing sufficient distancing of the molecules coupled to thecoupling agent, and its synthesis method is long. Also a stabilityproblem of the coupling agent may arise on account of the presence ofthe lysine —COOH function which could react with the alcoxyaminefunction known to be highly reactive.

The applicant has now developed novel coupling agents with which toovercome the disadvantages of prior art coupling agents in that theyallow sufficient distancing of the coupled molecules, they are highlysoluble owing to the nature of their spacer arm and the conjugatesobtained are very stable. In addition, the coupling agents of theinvention allow improved detection sensitivity for diagnostic tests.

Therefore, a first subject of the present invention consists of couplingagents having the following general formula (I):

in which:

-   -   Z represents —NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)—,    -   A represents a group chosen from among:    -   B represents a group chosen from among:    -   Y represents a group ending with —C(O)—NH₂ and which is inert        vis-à-vis the aminooxyalkylene function, the maleimide group or        groups of B and possibly A and vis-à-vis the pattern        —(CH₂)₂—O—(CH₂)₂—O—CH₂— of Z,    -   Z′ represents —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—,    -   n is an integer lying between 0 and 3,    -   m is an integer lying between 1 and 3,    -   o is an integer lying between 1 and 3,    -   p and p1 to p7 are each independently an integer lying between 1        and 4,    -   q and q1 are each independently an integer lying between 0 and        3,    -   r and r1 to r5 are each independently an integer lying between 2        and 5,    -   s is an integer of 0 or 1,    -   t is an integer lying between 1 and 7, and    -   u and u1 to u4 are each independently an integer lying between 2        and 10.

Another subject of the invention consists of activated intermediatesformed of a coupling agent such as defined above and of:

-   -   a molecule having on its surface at least one aldehyde and/or        ketone function before conjugation, or    -   one to eight molecules having on their surface at least one free        thiol function before conjugation.

A further subject of the invention consists of conjugates formed of acoupling agent such as defined above and linked firstly to a moleculehaving on its surface at least one aldehyde and/or ketone functionbefore conjugation, which is preferably a molecule of biologicalinterest, and secondly to one to eight molecules having on their surfaceat least one free thiol function before conjugation, which arepreferably markers.

A final subject of the invention consists of the use of a conjugate suchas defined above for in vitro diagnostic methods of diseases involvingrecognition of a ligand-anti-ligand pair.

The coupling agents of the invention are as defined in formula (I)above. They are characterized by the presence of:

-   -   an aminooxyalkylene function: NH₂—O—(CH₂)_(o)—, o being as        defined for (I), so that it can specifically bind with a        molecule having on its surface at least one aldehyde and/or        ketone function,    -   one to eight maleimide functions at B and optionally at A to be        able to bind specifically with one to eight molecules having on        their surface at least one free thiol function, and    -   a spacer arm Z of polyethyleneglycol type which is long and is        able to impart better solubility to the coupling agent.

Also, the association of the three aforesaid characteristics givesimproved stability to the conjugates formed from said coupling agents.

The coupling agents of the invention also contain a Y group that isinert towards the aminooxyalkylene function: NH₂—O—(CH₂)_(o)—, o beingas defined for (I), towards the maleimide group(s) of B and optionally Aand towards the —(CH₂)₂—O—(CH₂)₂—O—CH₂— pattern of Z, i.e. the Y groupis unable to react with these patterns or functions. In addition, Y endsin —C(O)—NH₂. This —C(O)—NH₂ ending is of importance as, unlike the—COOH function such as present in the coupling agent described inBioconjugate Chemical, 1994, 556, 636-646, it allows the avoidance ofany stability problem due to the presence of the aminooxyalkylene. Inaddition, the coupling agent so obtained does not have any ionic charge.According to a preferred embodiment, Y is —C(O)—NH₂.

The coupling agents of the invention may include one to eight maleimidefunctions allowing the attachment of one to eight molecules having ontheir surface at least one, generally several, free thiol functionsbefore conjugation, respectively.

It will be noted that

n is an integer between 0 and 3

for example, means that n may be 0, 1, 2 or 3.

In coupling agent (I), each Z or Z′ group is linked by its —NH— functionto a —C(O)— function and by its —C(O)— function to a —NH— function.

The coupling agents of the invention comprise at least one asymmetriccarbon and the different optical isomers form an integral part of theinvention. The subject of the present invention is therefore couplingagents of formula (I) in the form of pure isomers but also in the formof a racemic mixture or of any proportion. Coupling agents (I) areisolated in pure isomer form using conventional separation techniques,for example by chiral phase chromatography.

According to one preferred embodiment, the coupling agents of theinvention only have one maleimide function. In this case, s in formula(I) equals 0 and B preferably represents:

u being as previously defined.

When the coupling agents of the invention have more than one maleimidefunction, they may either be in dendrimer (or branched) form or inlinear form, depending upon the value of A and B.

According to a preferred embodiment, A and B are identical andrepresent:

Z′, p1 and u being as previously defined,

-   -   so that the coupling agents of the invention are in dendrimer        form and have two maleimide functions.

According to another preferred embodiment, A and B are identical andrepresent:

Z′, p1, p2, p3, q1, r1, u1 and u2 being as previously defined,

-   -   so that the coupling agents of the invention are in dendrimer        form and have four maleimide functions.

According to a further embodiment, A and B are identical and represent:

Z, Z′, p1 to p7, q1, r1 to r5 and u1 to u4 being as previously defined,

-   -   so that the coupling agents of the invention are in dendrimer        form and have eight maleimide functions.

When the coupling agents are in linear form, A represents:

and

-   B represents:-   Z′, t, p1 to p3, q1, r1 and u, u1 and u2 being as previously    defined,    and this forms another embodiment of the invention.

In the coupling agents of the invention, the coupling arm may be ofvarying length, but it must be sufficient to distance the moleculeswhich are to be coupled, in particular on account of their sterichindrance, and when the coupling agents have several maleimide functionswhich are to be coupled to as many molecules with free thiol functions.

The length of the arms varies in relation to the indices m, n, o, p, p1to p7, r, r1 to r5, q, q1 and u, u1 to u4.

According to one embodiment, the coupling agents of the invention meetat least one of the following conditions:

-   -   n equals 0,    -   m equals 1,    -   p and p1 to p7, when applicable, equal 2,    -   q and q1, when applicable, equal 0,    -   r and r1 to r5, when applicable, equal 4,    -   u and u1 to u4, when applicable, equal 2 and    -   o equals 1.

The preferred coupling agents are chosen from among the following:

-   -   compound of formula (I) in which m=1, Y=—C(O)—NH₂, O=1, n=0, p        2, s=0 and B represents:        and    -   compound of formula (I) in which m=1, Y=—C(O)—NH₂, o=1, n=0,        p=2, r=4, s=1, q=0 and A and B are identical and represent:        p1 equalling 2.

The coupling agents of the invention are obtained by solid phasesynthesis of peptide synthesis type either manually or preferablyautomatically on commercially available synthesizers such as the ABI 431A synthesizer.In other words, the coupling agents, which can be divided into thesynthons described below, which are amino acid or related synthons, areobtained from a synthesis support (solid phase) such as the commercialresin RINK-Amide-MBHA (NOVABIOCHEM), making possible the attachment ofthe C-terminal end of the first synthon (synthon I) of the couplingagent to be synthesized before proceeding with the synthesis of saidagent by gradually adding suitable synthons chosen from among synthonsII to IV:

where n, m, and o are as defined previously, q′ represents q or q1, r′represents r, r1, r2, r3, r4, or r5 and u′ represents u, u1, u2, u3, oru4 such as previously defined, Boc denotes tert-butyloxycarbonyl, Fmocdenotes fluorenylmethoxycarbonyl and Y′ corresponds to Y devoid of the—C(O)—NH₂ function.The coupling of a synthon IV with a synthon I is made after deprotectingthe Fmoc group.

The reagents used during the coupling cycles of the different synthonsare known to persons skilled in the art and are described for example inChemical Approaches to the Synthesis of Peptides & Proteins, Paul LloydWilliams, Fernando Albericio, Ernest Giralt, CRC Press. Aftersynthesizing the coupling agent of the invention, it is detached fromthe support following an operating mode known to those skilled in theart (Chemical Approaches to the Synthesis of Peptides & Proteins, supra)and is simultaneously deprotected (Boc group) in the presence of anacidolysis solution such as a solution containing trifluoroacetic acid.

The coupling agents are then purified, for example by semi-preparativeBECKMAN HPLC on a C₁₈ reverse phase column.

The coupling agents so obtained can be used to couple firstly themolecules having on their surface at least one aldehyde and/or ketonefunction before conjugation, which will react with the aminooxyalkylenefunction of the agents, and secondly those molecules having on theirsurface at least one free thiol function before conjugation which willreact with the maleimide function or functions of said agents.

The subject of the invention is therefore activated intermediates formedof a coupling agent of the invention with either a molecule having onits surface at least one aldehyde or ketone function before conjugation,or with one to eight molecules having on their surface at least one freethiol function before conjugation, preferably one to four of saidmolecules.

According to a preferred embodiment, the activated intermediates areformed of a coupling agent of the invention and of a molecule having onits surface at least one, and generally more than one, aldehyde and/orketone function before conjugation.

The molecules having aldehyde or ketone functions on their surfacecomprise all molecules having such functions or which are modified toinclude such functions. By way of example, peptides having an N-terminalserine or threonine may be cited, and molecules of biological interestsuch as antigens, antibodies, haptenes, glycoproteins, or any othermolecule able to bind with a linkage partner.

The modification of molecules desired to have aldehyde or ketonefunctions may be performed by reaction with reagents leading to suchfunctions, such as periodate.

As examples of molecules having aldehyde functions, periodate-oxidizedglycoproteins may be cited, such as gp160 the envelope glycoprotein ofthe HIV-1 virus, sugars. As examples of molecules having ketonefunctions, any molecule may be cited which contains a levulinyl (ketone)group.

According to one preferred embodiment, the molecule having aldehydefunctions on its surface is a glycoprotein oxidized with periodate andpreferably gp 160 so oxidized.

Persons skilled in the art may easily determine which molecules havefree thiol functions on their surface, either naturally or by chemicalreaction, before conjugation.

Examples of said molecules comprise markers, which form an embodiment ofthe invention.

By marker is meant any molecule able to generate a detectable signaleither directly or indirectly. A non-limitative list of these markersincludes:

-   -   enzymes which produce a detectable signal for example by        colorimetry, fluorescence, luminescence, such as horseradish        peroxidase, alkaline phosphatase, α-galactosidase,        glucose-6-phosphate dehydrogenase,    -   chromophores such as fluorescent, luminescent compounds, dyes,    -   radioactive molecules containing ³²P, ³⁵S or ¹²⁵I, and    -   fluorescent molecules,        on the understanding that these markers will be optionally        modified to have free thiol functions.

According to one embodiment of the invention, the marker is alkalinephosphatase, previously modified.

The modification of molecules desired to have free thiol functions maybe conducted by reaction with reagents leading to said functions such asTraut's reagent (Pierce) also called imminothiolane.

With alkaline phosphatase, modification is performed in the presence ofTRAUT's reagent (Pierce).

The quantity of marker to be used depends upon the number of maleimidefunctions contained in the coupling agent of the invention that is used.It may easily be determined by persons skilled in the art.

Evidently, the aforesaid examples, firstly in respect of moleculeshaving at least one aldehyde and/or ketone function, and secondly ofmolecules having at least one free thiol function, are not restrictiveand are interchangeable. In other words, for example, a coupling agentmay be coupled, via its maleimide function, with a molecule ofbiological interest modified to include at least one free thiolfunction, and, via its aminooxyalkylene function, with a marker modifiedto include at least one aldehyde and/or ketone function.

Both types of molecules having the functions described above linked tocoupling agents of the invention form conjugates which constitute afurther subject of the invention.

According to an embodiment of the invention, the conjugates of theinvention meet at least one of the following conditions:

-   -   the molecule having at least one aldehyde and/or ketone function        is a molecule of biological interest, in particular previously        oxidized glycoprotein gp 160, and    -   the molecule or molecules having at least one free thiol        function are markers, in particular previously modified alkaline        phosphatase.

The conjugates so obtained may be used for in vitro diagnosis methods ofdiseases involving recognition of a ligand/anti-ligand pair, themolecule of biological interest forming the ligand.

With the conjugates of the invention it is possible for example todetermine in a biological sample the presence of an anti-ligand whichwill attach itself to the ligand of said conjugate, such attachmentbeing detected by means of the marker of said conjugate.

Examples of ligand/anti-ligand pairs are well known to persons skilledin the art, which is the case for example for the following pairs:biotine/streptavidine, haptene/antibody, antigen/antibody,peptide/antibody, sugar/lectine, polynucleotide/complementarypolynucleotide, one of the elements of these pairs evidently forming themolecule of biological interest.

The in vitro diagnosis methods able to use the conjugates of theinvention are sandwich type methods in particular which are largelyknown to persons skilled in the art. As an example the ELISA method maybe cited, or the ELOSA method, ELISPOT method and Western-blot method.

Those diseases which may be diagnosed with the complexes of theinvention are unlimited and include all diseases detected by thepresence of a specific marker of the disease, of the type molecule ofbiological interest, for which a linkage partner exists. As an example,viral diseases such as AIDS may be cited, and solid cancers such asbreast or prostate cancer.

The coupling agents of the invention, such as defined previously, areable to improve the in vitro diagnosis of diseases by using directcoupling of a molecule of biological interest with one or more markers,with no intermediate biotinylation of said molecule of interest, owingto the length of their spacer arms and good stability of the formedconjugates. In addition, the use of the coupling agents of the inventionmakes it possible to amplify the detection of the ligand/anti-ligandbond since said coupling agents may bind with up to eight markers whilesolving the steric hindrance problem of a large number of molecules.

The invention will be more readily understood with the assistance ofappended FIG. 1, which shows the enzymatic tracing using a conjugate ofthe invention, and with the assistance of the following examples whichare given as non-restrictive examples.

EXAMPLES Example 1

Compound of formula (I) in which m=1, Y=—C(O)—NH₂, o=1, n=0, p=2, s=0and B represents:

282 μmoles of Fmoc-RINK-Amide-MBHA resin (Novabiochem) were placed inthe reactor of a 431 A automatic synthesizer (Applied Biosystems). Fourcartridges of the three synthons I, II and TV were prepared containing ⅓mmole of each synthon as follows:

-   -   one cartridge containing 166.4 mg (approx. 333 μmol) of the        following synthon (corresponding to synthon I):        where Boc denotes t-butyloxycarbonyl and Fmoc denotes        fluorenylmethoxycarbonyl,    -   two cartridges each containing 128.5 mg (approx. 333 μmol) of        synthon II:        in which Fmoc is as defined above, and:    -   one cartridge containing 56.4 mg (approx. 333 μmol) of synthon        IV:

The following coupling cycle was conducted four times with the aforesaidcartridges, in the following order:

-   -   rinsing and et solvation of the resin with N-methylpyrrolidinone        (NMP),    -   dual treatment, for approximately 4 min, then for more than 10        min, of synthon I (1^(st) passage) or of the synthon(s)/resin        complex (3^(rd) to 4^(th) passage) with a solution of piperidine        of approximately 40% in NMP to deprotect the Fmoc group,    -   simultaneously, dilution in a minimum amount of NMP and        preactivation of the synthon in its cartridge through the        addition of one equivalent of the coupling agent        hexafluorophosphatebenzotriazolyltetramethyluronium (HBTU) in        the form of a 0.2 M HBTU solution in N,N-dimethylformamide,    -   emptying the reactor and multiple rinsing of the resin with NMP,    -   transfer of the content of the synthon cartridge to the reactor        and addition to the reactor of approx. 2.5 equivalents of the        tertiary coupling base disopropylethylamine (DIEA) in the form        of a 1.67 M DIEA solution in NMP, and    -   coupling for one hour and reactor emptying.

Subsequently, a resin rinsing cycle was performed with several rinsingsin NMP, followed by dichloromethane (DCM).

Deprotection and detaching of the coupling agent was then conducted byleaving it to react at room temperature for one hour in approximately6.5 ml of trifluoroacetic acid solution (TFA) with 5% water, then bytransferring the liquid phase into a container containing approximately100 ml ethyl ether at −10° C. and rinsing the resin with approximately 4ml TFA and 4 ml DCM, these latter liquid phases having been added to thefirst in the ether container.

The container containing the coupling agent was centrifuged at 3500 rpmat −5° C. for 10 min and the ethereal supernatant was removed. Thisoperation was repeated twice after addition of ether.

After vacuum drying, 0.14 g of crude mixture was obtained.

The presence of the coupling agent was verified in the crude mixture bytaking up the mixture in MilliQ demineralized water (Waters System) andby analysing an aliquot quantity of this mixture by BECKMANN HPLC(reverse phase C₁₈ VYDAC chromatography column, eluant E_(A) solution ofMilliQ demineralized water with 0.1% TFA, eluant E_(B) solution of 95%acetonitrile and 5% MilliQ water with 0.1% TFA, purification rate 1ml/min) using a gradient of 0 to 80% acetonitrile over 30 minutes. Underthese conditions, the coupling agent has a retention time in the orderof 9.7 min (dead volume peak at around 3 min).

The coupling agent was purified with semi-preparative BECKMAN HPLC(reverse phase C₁₈ VYDAC chromatography column, eluant E_(A) solutionMilliQ demineralized water with 0.1% TFA, eluant E_(B) solution of 95%acetonitrile and 5% MilliQ water with 0.1% TFA, purification rate 22ml/min). The programmed gradient for this purification was as follows:

-   -   0% eluant E_(B) from t=0 to 10 min,    -   change to 5% eluant E_(B) from t=10 to 11 min and    -   change to 19% eluant E_(B) from t=11 to 41 min.

Fractions were taken as from t=16.5 min at the rate of 0.2 min per tubeand for 40 tubes. Isocratic analysis was made of tubes 11 to 40 for 10min with 11% eluant E_(B) on the analytical system.

The purest fractions were grouped together and freeze-dried in a VIRTISfreeze-drier. After freeze-drying and weighing, the product was taken upin MilliQ water and aliquoted in an Eppendorf tube.

Example 2

Compound of formula (I) in which: m=1, Y=—C(O)—NH₂, o=1, n=0, p=2, r=4,s=1, q=0 and A and B are identical and represent:

p1 equalling 2

To prepare the title compound, the operating mode described in example 1was repeated, with the sole exception that the starting product was 153μmol of reaction product between the resin, a cartridge of synthon I andtwo cartridges of synthon II such as described in example 1, and thefollowing synthon cartridges were used:

-   -   one cartridge containing approximately 166 μmol of the following        synthon (corresponding to synthon III:        in which Fmoc is such as defined above,    -   four cartridges each containing approximately 333 μmol of        synthon II:        in which Fmoc is such as defined above, and    -   two cartridges containing approximately 333 μmol of the        following synthon IV:

In this manner a product in gum form was obtained which was purified asindicated above except that the following gradient cycle was used:

-   -   10% eluant E_(B) from t=0 to 10 min,    -   change to 15% eluant E_(B) from t=10 to 11 min, and    -   change to 27% eluant E_(B) from t=11 to 41 min.

Example 3 Preparation of the Activated Intermediate gp 160/CouplingAgent of the Invention

The glycoprotein gp160 (ABL) was dialysed overnight at 2-8° C. against a50 mM acetate buffer pH 4.5 in the presence of 0.01% SDS. Theglycoprotein obtained was oxidized with 5 mM NaIO₄ in darkness for 15min at 18-25° C. The reaction was blocked with ethyleneglycol ( 1/200)and the oxidized protein was dialysed against a 25 mM solution of sodiumtetraborate pH 5.5.

A solution of the coupling agent was prepared as in example 2 with 2mg/ml in a sodium tetraborate solution.

The coupling agent solution was mixed with the oxidized glycoprotein tothe proportion of 20 mol coupling agent per 160 000 Da unit ofglycoprotein and was incubated for one hour at 18-25° C. under stirringto achieve coupling.

The mixture obtained was dialysed against a 50 mM phosphate buffer, 150mM NaCl, at pH 6.8.

Example 4 Preparation of the Conjugate gp160/Coupling Agent/AlkalinePhosphatase

The alkaline phosphatase was modified to include free thiol groups asfollows: the alkaline phosphatase (Biozyme) was dialysed against 10 mMphosphate buffer, 5 mM EDTA at pH 8. A solution of Traut's reagent(Pierce) was prepared with 3 mg/ml in 10 mM phosphate buffer, 5 mM EDTA,at pH 8 and left to incubate 2 hours at 18-25° C. The reaction mixturewas desalted on a Sephadex G-25 column (Pharmacia).

1 mg of activated intermediate obtained in example 3 was mixed with 3 mgmodified alkaline phosphatase and left to incubate overnight at 2-8° C.under stirring. The reaction was blocked (maleimide groups) through theaddition of a 10 mM β-mercaptoethanol solution to the buffer at pH 6.8and left to incubate 15 min at room temperature. The reaction wasblocked (residual thiol functions) with a solution of N-ethylmaleimide(Sigma) in the buffer at pH 6.8 and left to incubate 15 min at roomtemperature. Dialysis was then conducted for twice 45 min at roomtemperature and overnight at 2-8° C. against the PBS buffer.

The conjugate was then purified on the Superdex 200 chromatographycolumn (Pharmacia) (eluant: PBS, azide with 0.01% SDS, flow rate: 1ml/min, fractions: 1 ml).

The enzymatic activity of the chromatography fractions was examinedusing a microplate test consisting of sampling 25 μl of each fraction,making up to 100 μl with PBS, adding 100 μl pNPP, leaving to incubatefor 5 min, blocking the reaction with 20 μl 0.5 N NaOH and measuring theoptical density.

The tracing of the enzymatic activity of this test is given in thesingle FIGURE which evidences the good marking level obtained. Thedifferent fractions are indicated over the abscissa axis.

The optical densities of the conjugate alone at 280 nm (curve 1), and ofthe conjugate after enzymatic reaction at 405 nm (curve 2), weremeasured.

Example 5 Comparison of the Detection Sensitivity of a Conjugate of theInvention and of a Conjugate Obtained with a Pierce Coupling Agent

A conjugate obtained with the coupling agent of example 1 was used and aconjugate obtained with the Pierce coupling agent (EMCH, such asdescribed below) and the sensitivity was compared in terms of detectionachieved by these different conjugates.

For this purpose, in a reaction tank 150 μl of 1 μm magnetic particleswere added in a 1% suspension and diluted to 1/50 in a buffer (200 mMTris—150 mM NaCl pH 8.5—BSA 10 g/1-5% Tween 20, NaN₃ 0.9 g/l),functionalised with —COOH groups and sensitized with the gp160glycoprotein of the HIV-1 virus, and 150 μl of sample. The testedsamples were either a pool of negative HIV-1 (HIV-1−) sera, or samplescontaining the HIV-1 (HIV-1+) virus diluted in a pool of negative sera.They were left to incubate 15 min at 37° C.

Subsequently 150 μl of reagent were added containing the conjugatesgp160/coupling agent/alkaline phosphatase prepared following theoperating mode described in example 4 using the appropriate couplingagent and diluted to an approximate concentration of 2 μg/ml in a bufferof 100 mM Tris—300 mM NaCl pH 7.4—Mannitol 2.5 g/1—NaN₃ 0.9g/1—Régilait® milk 2.5 g/1—BSA 2.5 g/1-0.1 mM ZnCl₂-1 mM MgCl₂. This wasleft to incubate 15 min at 37° C.

Three washings were conducted with a washing solution formed of acitrate buffer pH 6 containing NaCl, a detergent, an anti-foaming agentand anti-microbial agent (MAGIA, Biotrol).

The luminescence substrate Lumiphos 530 (Lumigen) was added to thereaction tank and left to incubate for 15 min at 37° C.

Luminescence was read off in RLU units (Relative Luminescence Unit)using a H7155 photomultiplier (HAMAMATSU) and the luminescence ratio wascalculated between the samples containing the virus and those withoutthe virus.

The results are given in table 1 below. TABLE 1 Sensitivity testLuminescence RLU HIV-1 + HIV-1 + (1/2000) (1/300) Ratio Ratio ConjugateHIV-1 − a1 a2 a3 a2/a1 a3/a1 with example 1 1170 58342 3409 49.9 2.9with EMCH 8103 134104 10621 16.6 1.3 with EMCH 3731 61526 5033 16.5 1.35(factor 2dilution)

The ratios obtained with the conjugate of the invention and with aconjugate prepared with EMCH such as indicated above, clearly show theimprovement in detection sensitivity when a coupling agent of theinvention is used, whether or not dilution is extensive (last 2 columnsin the table).

It is to be noted that, to obtain a control detection level (backgroundnoise) comparable with the level obtained with the conjugate of theinvention, the conjugate prepared from EMCH was diluted by a factor of 2(last line). It was therefore possible to verify that, with comparablebackground noise, the luminescence ratio remains low for the conjugateprepared from the coupling agent of the prior art.

Example 6 Comparison of Stability Between a Conjugate of the Inventionand a Conjugate Obtained with the EMCH Coupling Agent

To study the stability of the conjugates, the diluted conjugates wereleft to age as indicated in example 5, in an oven at 37° C. 8, 15 and 30days and their behaviour was compared with the same preparations storedat 4° C. The percentage results are given in table 2 below. TABLE 2Stability test 8 days 15 days 30 days at + 37° C. at + 37° C. at + 37°C. Conjugate of −8.7% −13.2% −37.1% the invention Conjugate −36.7%−57.3% −56.8% with EMCH

The results of table 2 clearly show that under forced aging conditions,the conjugate prepared with the coupling agent of the invention is muchmore stable than the conjugate prepared with a coupling agent of theprior art.

Example 7 Preparation of the Coupling Agent Described by Mikolajczvk S.et al. (Supra)

All preparative operations were conducted on a 431 A automatic peptidesynthesizer (APPLIED BIOSYSTEMS) at room temperature, in accordance withthe following schema:

Approximately 0.68 mole (in chloride equivalent) of highly acid labile2-chlorotrityl chloride resin (Novabiotech) were placed in the reactor.

Using a semi-automatic protocol, this resin was functionalised throughthe addition of a solution containing 1 mmole (approx. 535 mg)Fmoc-Lys(Dde)-OH (Dde: (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl,NOVABIOCHEM) and 1 mmole diisopropylethylamine (DIEA, ALDRICH) in 6 mldichloromethane (DCM, ABI) to fix the lysine protected on the resin by achemical bond of ester type.

After vortexing for 35 minutes, 0.5 ml anhydrous methanol (MERCK) wereinjected into the reactor with 5 ml of a 5M solution of 2.5 mmoles DIEAin N-methylpyrrolidone (NMP, ABI).

After washing the resin several times with DCM, a 20-30% piperidinesolution (ALDRICH) was caused to react under stirring in NMP for 2 times5 minutes. The resin was then rinsed several times with with NMP andDCM.

A solution was placed on the resin containing 6 ml DCM and approximately0.69 mmole Boc-Aoa-Osu and 0.25 ml of the 5M solution of 1.25 mmole DIEAin NMP already described above. The coupling reaction lasted 55 minutesunder stirring. The resin was then rinsed several times with NMP.

To perform selective deprotection of the Dde group, the resin wastreated 6 times 3 minutes with 8 ml of a 2% solution of hydrazinehydrate (ALDRICH) in dimethylformamide (DMF ALDRICH). Several rinsingswere then conducted with NMP.

To couple N-maleoyl-β-alanine, 0.6 mmole of N-maleoyl-β-alanine (FLUKA),i.e. approx. 100 mg taken up in approx. 5 ml NMP, were left to react onthe resin with 1 ml of a 0.6M solution of 1 equivalent HBTU couplingagent (QUANTUM APPLIGENE) in the DMF and 300 μl of 5M DIEA solution of2.5 equivalents of DIEA in the NMP. Coupling under stirring lastedaround 40 minutes, after which the resin was rinsed several times withNMP and then with DCM.

The coupling agent was detached from the resin by mild acidolysis on theresin treated at room temperature 9 times 2 minutes with 5.5 ml of a 1%solution of trifluroacetic acid (TFA, ACROS) in DCM. Each of thesevolumes was collected and they were added to 80 ml diethyl ether (SDS)previously cooled to −10° C. The ethereal mixture was evaporated in arotary evaporator (BÜCHI type) in fractions of a few dozen ml added to avolume of 50 ml cyclohexane (PROLABO) to remove the ether, the DCM, TFAand cyclohexane. Product recovery was 0.14 g, which represents a grossyield of 44%.

The coupling agent was purified and analyzed as follows:

All the product so obtained was slowly taken up in 2 ml acetonitrile(ACN, MERCK). When solubilisation was complete, 1 ml demineralized waterwas added and the solution transferred to a sampling bottle. The flaskwas rinsed with a mixture of 2 ml ethanol and 1 ml water which wereadded to the first collection volume (3 ml). This operation was repeatedwith water to finally obtain the coupling agent in approximately 50 mlof clear mixture containing 4% ACN and 4% ethanol. This volume wasinjected in several injections into a BECKMAN semi-preparative highperformance liquid chromatography column (fitted with model 126 pumpsand 166 UV detector), with VYDAC reverse phase C₁₈ column (reference218TP152022) at a flow rate of 22 ml.min⁻¹. The working wavelength was214 nm. The 2 eluants used for this purification were A)

milliQ

demineralized water (WATERS) with 0.1% TFA and B) a 95% ACN—5% watermixture with 0.1% TFA.

The column was maintained at 10% of B) for 10 minutes to allow theinjection of the solution to be purified, then increased to 20% of B)over 1 minute thereafter continuing with a flat gradient of 20 to 31% B)over 30 minutes. At the column outlet 12 fractions were collected asfrom 19.3 minutes at the rate of 0.25 minute per tube.

An aliquot of each fraction was analyzed by BECKMAN analytical HPLC withthe same eluants as previously, but on a smaller reverse phase C₁₈ VYDACcolumn (reference 218TP54), with a flow rate of 1 ml.min⁻¹ and aconstant (isocratic) proportion of eluant B) of 26% over 10 minutes. Thefractions found to be the purest were grouped together and freeze-driedto obtain 12.6 mg of product, i.e. an apparent yield before final assayof the coupling agent of 2.7% since the start of synthesis. The couplingagent was taken up in a 50% ACN aqueous solution and aliquoted intofractions of 580 μg each (500 μg theoretical of which it was verifiedthat these fractions were 580 μg), which were freeze-dried. Thestructure of the protected coupling agent was confirmed by PE-SCIEX massspectrometry (API100 model) in which M=470.4 daltons (averagetheoretical 470.48 and mono-isotopic 470.20).

The coupling agent was deprotected by causing one of the 580 figaliquots to react with 100 μl TFA under stirring for 30 minutes at roomtemperature. This solution was then transferred to a 2 ml glassrecipient adapted to the centrifuger and to which 3 rinsings of 100 μlDCM were added. Evaporation was performed in a SPEED VAC® vacuumcentrifuger for 15 minutes. This yielded 580 μg coupling agent, i.e. ayield of 4.6%.

Example 8 Comparison of the Detection Sensitivity of a Conjugate of theInvention and a Conjugate Obtained with the Coupling Agent Obtained inExample 7

Conjugates of gp 160/coupling agent/alkaline phosphatase were prepared fallowing the operating mode indicated in examples 3 and 4, with thecoupling agent of the invention described in example 1 and with thecoupling agent of Mikolajczyk S. et al. such as described in example 7,with the exception that for the prior art coupling agent:

-   i) compared with example 7 deprotection was conducted as follows: a    freeze-dried aliquot of the coupling agent (580 μg) was taken and    left to react with 100 μl trifluoroacetic acid for 30 minutes at    room temperature and under stirring. This reaction mixture was then    transferred to a flask compatible with the    SPEED VAC    vacuum centrifuger and to which were added the 2 dichloromethane    rinsing products (2×100 μl). Drying was conducted for 20 minutes on    this apparatus (without heating the chamber) to obtain a slightly    yellow residue of said coupling agent,-   ii) for the oxidation step of gp160 (example 3), a 0.2 M solution of    NaIO₄ was used and it was blocked with a 1/150 solution of    ethyleneglycol.

To determine detection sensitivity, the operating mode described inexample 5 was used, with the exception that 250 μl of magnetic particleswere used and a conjugate solution concentration of 0.5 μg.ml¹, as wellas sera with a scarcely positive HIV-1 content (HIV-1+low), fairlypositive HIV-1 content (VIH-1+average) and highly positive HIV-1 content(VIH-1+high).

By scarcely positive sera is meant sera scarcely rich in anti-HIVantibodies, i.e. whose detection measurement is close to threshold whenanalysed with a commercial kit. By fairly positive sera is meant serafairly rich in antibodies and by highly positive sera is meant serahighly rich in antibodies.

Luminescence in RLU units and the luminescence ratios obtained with thetwo compounds are given in table 3 below. TABLE 3 Luminescence RLUHIV-1 + low VIH-1 + average VIH-1 + high Ratio Ratio Ratio ConjugateHIV-1 − a1 a2 a3 a4 a2/a1 a3/a1 a4/a1 with example 1 1291 18258 99444655177 14.14 77.01 507.36 with example 7 2386 15688 90593 517137 6.5737.97 216.74

Here again, the ratios obtained with the conjugate of the invention andwith a conjugate prepared with the coupling agent of Mikolajczyk S. etal. such as indicated above, clearly show the improvement in detectionsensitivity when the coupling agent of the invention is used.

1. Coupling agent having the following general formula:

in which: Z represents —NH—(CH₂)₂—O—(CH₂)₂—O—CH₂—C(O)— A represents agroup chosen from among:

B represents a group chosen from among:

Y represents a group ending in —C(O)—NH₂ and which is inert vis-à-visthe aminooxyalkylene function, the maleimide group or groups of B andoptionally of A and vis-à-vis the pattern —(CH₂)₂—O—(CH₂)₂—O—CH₂— of Z,Z′ represents —C(O)—CH₂—O—(CH₂)₂—O—(CH₂)₂—NH—, n is an integer lyingbetween 0 and 3, m is an integer lying between 1 and 3, o is an integerlying between 1 and 3, p and p1 to p7 are each independently an integerlying between 1 and 4, q and q1 are each independently an integer lyingbetween 0 and 3, r and r1 to r5 are each independently an integer lyingbetween 2 and 5, s is an integer of 0 or 1, t is an integer lyingbetween 1 and 7, and u and u1 to u4 are each independently an integerlying between 2 and
 10. 2. Coupling agent as in claim 1, characterizedin that Y is —C(O)—NH₂.
 3. Coupling agent as in claim 1, characterizedin that s equals
 0. 4. Coupling agent as in claim 3, characterized inthat B represents:

u being such as defined in claim
 1. 5. Coupling agent as in claim 1,characterized in that A and B represent:

Z′, p1 and u being such as defined in claim
 1. 6. Coupling agent as inclaim 1, characterized in that A and B represent:

Z′, p1, p2, p3, q1, r1, u1 and u2 being such as defined in claim
 1. 7.Coupling agent as in claim 1, characterized in that A and B represent:

Z, Z′, p1 to p7, q1, r1 to r5 and u1 to u4 being such as defined inclaim
 1. 8. Coupling agent as in either of claims 1 or 2 claim 1,characterized in that A represents:

and B represents:

Z′, t, p1 to p3, q1, r1, u, u1 and u2 being such as defined in claim 1.9. Coupling agent as in claim 1, characterized in that n equals
 0. 10.Coupling agent as in claim 1, characterized in that m equals
 1. 11.Coupling agent as in claim 1, characterized in that p and p1 to p7, whenapplicable, equal
 2. 12. Coupling agent as in claim 1, characterized inthat q and q1, when applicable, equal
 0. 13. Coupling agent as in claim1, characterized in that r and r1 to r5, when applicable, equal
 4. 14.Coupling agent as in claim 1, characterized in that u and u1 to u4, whenapplicable, equal
 2. 15. Coupling agent as in claim 1, characterized inthat o equals
 1. 16. Coupling agent as in claim 1, characterized in thatit is: the compound of formula (I) in which m=1, Y=—C(O)—NH₂, o=1, n=0,p=2, s=0 and B represents:

or the compound of formula (I) in which m=1, Y=—C(O)—NH₂, O=1, n=0, p=2,r=4, s=1, q=0 and A and B are identical and represent:

p1 equalling
 2. 17. Activated intermediate formed of a coupling agent asin a claim 1 and of a molecule having on its surface at least onealdehyde and/or ketone function before conjugation.
 18. Activatedintermediate as in claim 17, characterized in that the molecule havingon its surface at least one aldehyde function is a glycoprotein oxidizedwith periodate.
 19. Activated intermediate as in claim 18, characterizedin that said glycoprotein is the gp160 glycoprotein.
 20. Activatedintermediate consisting of a coupling agent as in claim 1 and of one toeight molecules having on their surface at least one free thiol functionbefore conjugation.
 21. Activated intermediate as in claim 20,characterized in that it contains from one to four molecules having ontheir surface at least one free thiol function before conjugation. 22.Activated intermediate as in claim 20, characterized in that said othermolecule(s) having on their surface at least one free thiol function is(are) a marker.
 23. Activated intermediate as in claim 22, characterizedin that the marker is alkaline phosphatase previously modified toinclude free thiol functions.
 24. Conjugate formed of a coupling agentas in claim 1 linked to a molecule having on its surface at least onealdehyde and/or ketone function before conjugation, and to one to eightmolecules having on their surface at least one free thiol functionbefore conjugation.
 25. Conjugate as in claim 24, characterized in thatthe molecule having on its surface at least one aldehyde and/or ketonefunction before conjugation is a molecule of biological interest. 26.Conjugate as in claim 25, characterized in that the molecule of interestis the gp160 glycoprotein previously oxidized.
 27. Conjugate as in claim24, characterized in that the molecule or molecules having on theirsurface at least one free thiol function before conjugation are markers.28. Conjugate as in claim 27, characterized in that the markers arealkaline phosphatase previously modified to include free thiolfunctions.
 29. Use of a conjugate as in claim 24 for in vitro diagnosticmethods of diseases involving recognition of a ligand-anti-ligand pair.